This proposal is designed to provide insights into the implementation of motivated behaviors by specific neural circuits, by testing and applying new tools that complement the control capability of optogenetics with novel structural and activity insights. Integrating our new chemical engineering- based technology (HETT: hydrogel-electrophoretic tissue transmutation) with imaging, behavioral, and optogenetic analysis, we propose to connect the wiring to the activity and causal impact of cell populations involved in mammalian motivated behavior, with simultaneous molecular precision and global scope. In brief, the opportunity has now emerged to measure, in models of mouse behavior relevant to motivation-linked clinical conditions: 1) brainwide neuronal activity patterns during specific motivated behaviors, 2) immunophenotypes of those activated cells, 3) global projection patterns of those same cells, and 4) causal impact of those cells on circuit activity and behavior. In Aim 1, we employ HETT to rapidly transform dense intact brain tissue into transparent and antibody-permeable form, and thereby rapidly and efficiently map in global (brainwide) fashion those neural populations with altered activity in settings of motivated behaviors (conditioned place preference, social interaction, sucrose consumption, and escape behavior); we have recently shown that in all of these behaviors we are able to bidirectionally modulate the key behavioral outcome with corresponding bidirectional modulation of dopamine neurons, providing substantial experimental leverage for Aims 2 and 3 below. The resulting brainwide activity maps will be made available online in rendered volumes as a community resource. In Aim 2, building on both our preliminary data as well as new data from Aim 1, in neural populations specifically and bidirectionally modulated during the four dopamine neuron-driven behaviors, we will bring to bear causal sufficiency and necessity tests using optogenetics in freely-moving mice to assess for modified behavior as a result of controlling activity in the implicated projections and populations of cells downstream of the dopamine neurons. We will track not only behavior but also local circuit activity in freely-moving mice, and HETT analysis will record on an individual-animal basis, linked to behavior, the extent and nature of circuit influences exerted in each experimental subject as well as the brainwide projection patterns of the causally implicated cell populations. Finally, in Aim 3 we seek circuit-dynamics insights into motivated behavior, applying high-speed volumetric activity readouts both in vivo and in acute slices from target brain regions implicated in Aims 1 and 2, during optogenetic drive of the dopamine neurons. The imaged tissues will then be processed for HETT, thereby transforming the very same 1) behaviorally tested and 2) live-imaged tissue into 3) a rigid and stable structure that can be interrogated for wiring and immunophenotype. Together, the approaches proposed here will integrate novel technology to probe fundamental causal underpinnings and mechanisms of circuit activity controlling motivated behaviors in freely-moving mammals.